Receptor tyrosine kinases (RTKs) drive development and progression of many types of cancer. The archetypic member of the RTK family, epidermal growth factor receptor (EGFR), is overexpressed or mutated in bladder, brain, intestinal, colon, ovarian, lung and head-and-neck cancer. EGFR has become the major prognostic marker and therapeutic target in cancer. However, despite the success of EGFR targeted therapy in a subset of cancers expressing constitutively-active mutants of EGFR, EGFR inhibitors have not been effective in cancers expressing wild-type EGFR. This is attributable in large part to insufficient mechanistic understanding of the regulation of oncogenic signaling networks involving EGFR in tumors in vivo. The long-term objective of our research is to elucidate regulatory mechanisms of EGFR signaling and subsequently contribute to addressing the important clinical question of how to improve EGFR targeted therapy. We propose to accomplish this goal by defining the mechanisms by which EGFR signaling is regulated by endocytosis focusing on EGFR overexpressing head-and- neck squamous cell carcinoma (HNSCC) as the main experimental model. Our research aims at testing two broad hypotheses: 1) dysregulation of EGFR endocytosis is critical to cancer cell growth, survival and motility; and 2) endocytic system can be explored to develop new predictive/prognostic markers and identify new cancer therapeutic targets. Our studies in cultured cells during last years established working models of key stages of EGFR endocytic trafficking in vitro. We have also developed approaches to analyze EGFR activities and endocytosis in vivo in mouse tumor xenografts. Our analysis of the EGFR-dependent phosphoproteome using time-resolved multiplexed mass-spectrometry identified new putative signaling pathways initiated by EGFR in endosomes. We are now in a unique position to 1) elucidate the mechanisms of endocytic trafficking of EGFR in vivo in EGFR-dependent tumor models; 2) define the major signaling pathways involved in growth and motility of cancer cells that are triggered by endosomal EGFR; 3) decipher the complex effects of dysregulated EGFR endocytic trafficking on tumorigenic processes in mouse tumor models; and 4) test the hypothesis that slow EGFR endocytosis correlates with the sensitivity of human HNSCC to the therapeutic EGFR antibody, cetuximab. The proposed studies will untangle the multi- faceted regulatory mechanisms underlying the impact of dysregulated EGFR endocytic trafficking on signaling processes in tumor cells in vitro and in vivo, and develop strategies for using the components of the EGFR/RTK endocytic machinery as prognostic markers and therapeutic targets in cancer.